The subject of the present invention is novel TGF-β-enriched milk protein fractions, a process for their preparation and their use for the preparation of a medicament and/or a food composition intended for the prevention and/or treatment of chronic inflammatory disease, and in particular of psoriasis.
Psoriasis is a chronic dermatological condition characterized by an erythemato-squamous eruption, which evolves by flare-ups, predominantly on the elbows, the knees and the scalp.
From a biological point of view, psoriasis is a chronic inflammatory process which is characterized by abnormal proliferation and differentiation of the keratinocytes, associated with an infiltration of the dermis and of the epidermis by T lymphocytes and polynuclear neutrophils which form micro-abscesses in the horny layer.
The causes for the appearance of psoriasis in an individual are poorly known. Several factors which promote the appearance of psoriasis are cited:                a hereditary factor,        a psychological factor (stress, hormonal changes, . . . ),        an immune abnormality.        
Finally, certain medicaments and bacterial or viral infections are capable of triggering psoriasis attacks.
Currently, no treatment is capable of curing psoriasis. The treatments known up until now can merely postpone and/or attenuate the symptoms of psoriasis.
For psoriasis limited to a few plaques, vitamin D is prescribed, optionally combined with corticoids applied locally. It is also possible to prescribe retinoids applied topically.
In the most serious cases, phototherapy is prescribed, or optionally methotrexate or retinoids by the general route. The latter treatments are associated with major side effects.
No satisfactory treatment therefore currently exists for psoriasis.
The other chronic inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, Crohn's disease, multiple sclerosis, lupus erythematosus, pose the same problems for the practitioner: no treatment exists which makes it possible to cure these pathological conditions and the existing treatments for treating the manifestations of these pathological conditions are either inadequate or associated with very severe side effects. A chronic inflammatory component is also present in autoimmune diseases.
It is known that growth factors, like chemokines and cytokines, produce effects on the inflammatory processes. These effects range from the amplification to the suppression of the inflammatory process.
It is known in particular that certain growth factors in milk have a modulatory activity on the inflammatory process.
For example, the document WO 96/34614 describes the use of a milk product for the preparation of a medicament for the prevention of a lesion of the mucous membrane of the digestive tube caused by a chemotherapy or a radiotherapy.
The extract is preferably obtained by passing milk over a cation-exchange chromatography column. It preferably comprises lactoferrin and/or lactoperoxidase and a growth factor, in particular TGF-β.
TGF-β (Transforming Growth Factor) is a peptide growth factor which regulates cell growth and differentiation. It is a dimeric molecule of 25 kD which may exist in various isoforms: TGFβ1, TGFβ2 and TGFβ3. TGF-β is known for its capacity to regulate, and in particular to reduce certain stages of the immune and inflammatory reaction (G. Prud'homme et al., J. Autoimmun. (2000), 14, 23-42; M. Shull et al., Nature (1992), 359, 693-699; J. Graycar et al., Mol. Endocrinol. (1989), 3, 1977-1986; J. Letterio et al., Annu. Rev. Immunol. (1998), 16, 137-161). It exists in a latent form (non-biologically active) or in an active form.
It is present in milk in a very small quantity (30 μg/l of milk). However, numerous other factors are present in milk which have an activity different from that of TGF-β, possibly opposed to that of TGF-β, and the consequence thereof is that the TGF-β contained in milk has no notable action on the inflammatory process.
Moreover, the isolation of TGF-β from milk is barely feasible on an industrial scale, first of all because of cost considerations and also because the processes for isolation often cause denaturation of the target proteins and thereby lead to the production of a milk protein fraction which has lost all the biological properties of the proteins which it contains.
The document EP 0 313 515 describes a process for isolating TGF-β from milk. However, this process comprises a very large number of stages, of a complex nature, and therefore remains hardly viable from an economic point of view.
With the aim of obtaining a milk protein fraction which shows a biological activity on inflammatory processes, that is to say a reduction or a suppression of inflammatory processes, it would be necessary to be able, starting with milk, to increase the TGF-β content relative to the other proteins, while removing at least some of the antagonist factors for TGF-β which block its anti-inflammatory properties when it is in milk. The difficulty of this approach is all the more great since the precise antagonists of TGF-β are unknown.
Several authors have focused on the problem of the isolation of TGF-β-enriched milk protein fractions. That is the case for patent applications and patents WO 96/34614, EP 0 545 946, WO 01/25276, WO 03/008447, FR 2 827 240, EP 0 869 134.
The document EP-0 545 946 describes a process for extracting growth factors from whey; this process comprising the steps of: filtering the milk serum in order to remove the insoluble matter, adjusting the pH between 6.5 and 8, using a Sepharose-type cation-exchange resin onto which the main basic constituents are adsorbed and the main acid proteins are eluted, equilibrating the resin with a buffer, applying the filtrate onto the resin, eluting with a buffer with a high ionic strength, filtering in order to remove the salts, concentrating.
The document EP 0 869 134 describes a process for recovering one or more growth factors from milk or a milk derivative by adsorption onto a cation exchanger, fractional elution, production of a fraction enriched with growth factors, and then treating at a pH ranging from 3.5 to 4.5. The process is preferably applied with a very high superficial speed and a high liquid load.
The document WO 01/25276 describes a process for extracting TGF-β and IGF-1 comprising the following steps: recovering a basic fraction from milk by cation-exchange chromatography, passing the recovered fractions over a hydroxyapatite column, eluting with at least two eluants with increasing salt concentration so as to obtain two fractions:                an IGF-1-enriched fraction with IGF-1/TGF-β>10        a TGF-β-enriched fraction with TGF-β/IGF-1>5 and which contains from 30 to 50% of immunoglobulins relative to the total quantity of proteins.        
The document FR 2 827 240 describes a process for producing a TGF-β-enriched protein fraction in activated form, from a solution rich in soluble proteins of the aqueous phase of milk by: a) adjusting the soluble protein concentration to 5-30 g/l, b) precipitating by acid treatment, c) microfiltration-diafiltration, d) recovering the microfiltration retentate, e) drying.
The document WO 03/008447 describes a process for isolating the growth factors TGF-β, IGF-1, lacto-peroxidase and immunoglobulins. A basic protein fraction in milk is recovered by cation-exchange chromatography. The fraction obtained is passed over a hydrophobic interaction chromatography.
However, none of these processes has made it possible to obtain, under industrially viable conditions, a milk protein fraction having a real efficacy on chronic inflammatory pathological conditions and on their manifestations or symptoms, and in particular on psoriasis.